e Colistin is one of the few agents that retain activity against extensively drug-resistant strains of Klebsiella pneumoniae producing KPC-type carbapenemases (KPC-KP). However, resistance to colistin is increasingly reported among KPC-KP. Comparative genomic analysis of a pair of sequential KPC-KP isolates from the same patient including a colistin-susceptible isolate (KKBO-1) and a colistin-resistant isolate (KKBO-4) selected after colistin exposure revealed that insertional inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, is a genetic mechanism for acquired colistin resistance. The role of mgrB inactivation in acquired colistin resistance was confirmed by complementation experiments with wild-type mgrB, which restored colistin susceptibility in KKBO-4, and by construction of an mgrB deletion mutant from KKBO-1, which exhibited a colistin-resistant phenotype. Insertional mgrB inactivation was also detected in 60% of colistin-resistant mutants selected from KKBO-1 in vitro, following plating on colistin-containing medium, confirming the role (although not unique) of this mechanism in the emergence of acquired colistin resistance. In colistin-resistant mutants carrying insertional inactivation or deletion of the mgrB gene, upregulated transcription of phoP, phoQ, and pmrK (which is part of the pmrHFIJKLM operon) was detected. These findings confirmed the MgrB regulatory role in K. pneumoniae and were in agreement with the known association between upregulation of the PhoQ/PhoP system and activation of the pmrHFIJKLM operon, which eventually leads to resistance to polymyxins by modification of the lipopolysaccharide target.

e Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP) are challenging multidrug-resistant pathogens due to their extensively drug-resistant phenotypes and potential for epidemic dissemination in health care settings. Colistin is a key component of the combination antimicrobial regimens used for treatment of severe KPC-KP infections. We previously reported that insertional inactivation of the mgrB gene, encoding a negative-feedback regulator of the PhoQ-PhoP signaling system, can be responsible for colistin resistance in KPC-KP, due to the resulting upregulation of the Pmr lipopolysaccharide modification system. In this work we investigated the status of the mgrB gene in a collection of 66 colistin-resistant nonreplicate clinical strains of KPC-KP isolated from different hospitals in Italy and Greece. Overall, 35 strains (53%) exhibited alterations of the mgrB gene, including insertions of different types of mobile elements (IS5-like, IS1F-like, or ISKpn14), nonsilent point mutations, and small intragenic deletions. Four additional strains had a larger deletion of the mgrB locus, while the remaining 27 strains (41%) did not show mgrB alterations. Transcriptional upregulation of the phoQ and pmrK genes (part of the phoPQ and pmrHFIJKLM operon, respectively) was observed in all strains with mgrB alterations. Complementation experiments with a wild-type mgrB gene restored colistin susceptibility and basal expression levels of phoQ and pmrK genes in strains carrying different types of mgrB alterations. The present results suggest that mgrB alteration can be a common mechanism of colistin resistance among KPC-KP in the clinical setting.

A novel mcr variant, named mcr-1.2, encoding a Gln 3 -to-Leu functional variant of MCR-1, was detected in a KPC-3-producing ST512 Klebsiella pneumoniae isolate collected in Italy from a surveillance rectal swab from a leukemic child. The mcr-1.2 gene was carried on a transferable IncX4 plasmid whose structure was very similar to that of mcr-1-bearing plasmids previously found in Escherichia coli and K. pneumoniae strains from geographically distant sites (Estonia, China, and South Africa).

e Colistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceae producing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrA and of pmrK, which is part of the pmrHFIJKLM operon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrB in KPB-2 restored colistin susceptibility and reduced the transcription of pmrA and pmrK to basal levels, while expression of PmrB L82R in KPB-1 did not alter colistin susceptibility or upregulate pmrA and pmrK expression, confirming the dominance of wild-type PmrB versus the PmrB L82R mutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.

P olymyxins are old antibiotics that have recently regained popularity for treatment of severe infections caused by extensively drug-resistant (XDR) Gram-negative bacterial strains (1). As a likely consequence, emergence of polymyxin resistance is being increasingly reported in the clinical setting, especially among carbapenem-resistant Klebsiella pneumoniae isolates (2, 3). Acquired resistance to polymyxins is generally associated with chromosomal mutations (4, 5), but a new plasmid-mediated transferable resistance determinant, the mcr-1 gene, encoding a phosphoethanolamine transferase, has been described recently (6). The mcr-1 gene was originally detected in Enterobacteriaceae (mostly Escherichia coli) of animal and human origin in China (6) and subsequently also elsewhere (7-15), suggesting a broader distribution. In this communication, we report on the first detection of mcr-1 in colistin-resistant (COL-R) E. coli isolates from Italy. A retrospective analysis of the laboratory databases from the clinical microbiology laboratories of two Italian hospitals (Florence, central Italy; Lecco, northern Italy) revealed overall stable and low rates of colistin resistance among E. coli clinical isolates in the period 2012 to 2015 (Table 1). In both laboratories, routine susceptibility testing had been performed with the Vitek 2 system (bioMérieux, Marcy l'Etoile, France), and interpretation had been performed according to the EUCAST breakpoints (www.eucast .org). Eleven isolates that had been reported as colistin resistant (COL-R) were available for investigation, and nine of them were confirmed to be COL-R by reference broth microdilution (16). The COL MIC was 8 g/ml in all cases. The presence of mcr-1 was screened for by PCR and sequencing as previously described (6), using also an additional pair of primers (CLR5-F1, 5=-ATGATGCAGCATACTTCTGTGTGG; CLR5-R1, 5=-TCAGCGGATGAATGCGGTGC) targeting the extremities of the mcr-1 gene. Multilocus sequence typing (MLST) was carried out as described previously (17). Eight of the nine COL-R E. coli isolates were positive for the mcr-1 gene. In the sequenced region (positions 25 to 1576), with reference to the mcr-1 coding sequence (accession no. KP347127), the nucleotide sequences from all isolates were identical to that previously reported (6). Isolates positive for mcr-1 were detected from both centers, from inpatients in different wards, and also from two outpatients. Two mcr-1-positive isolates (LC-902/14